• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a substrate, p-Nitrophenyl phosphate (pNPP). The specific activity is >65,000 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Mouse myeloma cell line, NS0-derived Ala22-Pro320, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala22

• Predicted Molecular Mass

  34 kDa

• SDS-PAGE

  36-40 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM NaOAc, pH 5.0

• Recombinant Human TRACP/PAP/ACP5 (rhACP5) (Catalog # 3948-AP)

• Substrate: p-Nitrophenyl phosphate (Sigma-Aldrich, Catalog # N2765)

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

• NaOH, 0.2 M in deionized water

1. Dilute rhACP5 to 0.1 μg/mL in Assay Buffer.

2. Dilute Substrate to 2 mM in Assay Buffer.

3. In a plate combine 50 μL of rhACP5 and 50 μL of 2 mM Substrate. Include a Substrate Blank containing 50 μL Assay Buffer and 50 μL of 2 mM Substrate.

4. Incubate reaction at room temperature for 10 minutes in the dark.

5. Add 100 μL of 0.2 M NaOH to stop the reaction and develop the color.

6. Read (top read) absorbance in endpoint mode at 410 nm.

7. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard p-Nitrophenol (Sigma-Aldrich, Catalog # 241326).

Per Well:

• rhACP5: 0.005 μg

• Substrate: 0.5 mM

Background: TRACP/PAP/ACP5

Encoded by the ACP5 gene, Tartrate Resistant Acid Phosphatase (TRACP or TRAP) is also known as Purple Acid Phosphatase (PAP) or Acid Phosphatase 5 (ACP5) (1). The deuced amino acid (aa) sequence of human ACP5 predicts a signal peptide (aa 1 to 21) and a mature chain (aa 22 to 325). R&D Systems’ recombinant human ACP5 consists of aa 22 to 320, without the last 5 residues, RRARP.

ACP5 is expressed at high levels by osteoclasts, macrophages and dendritic cells (2). Two forms, 5a and 5b, circulating in human blood, are derived from different cell types and have different functions. Derived from macrophages and dendritic cells, 5a is a marker of inflammatory conditions. Derived from osteoclasts, 5b is a marker of bone resorption. Compared to 5a, 5b does not contain sialic acid residues, has a higher specific activity and pH optimum, and may be processed into a disulfide-linked dimer (3).

• References:

1. Janckila, A.J. and J.M. Halleen (2003) J. Bone Miner. Res. 18:1892.

2. Halleen, J.M. et al. (2006) Clin. Lab. 52:499.

3. Janckila, A.J. et al. (2003) J. Bone Miner. Res. 18:1916.

• Long Name:

  Tartrate Resistant Acid Phosphatase 5

• Entrez Gene IDs:

  54 (Human); 11433 (Mouse); 25732 (Rat)

• Alternate Names:

  ACP5; TRACP; TTRRAP